Abstract 1411: Doxorubicin Triggers Replicative Senescence and Death of Cardiac Progenitor Cells

Abstract

Doxorubicin (DOXO) is a very effective drug for the treatment of neoplastic diseases. However, DOXO causes a cardiomyopathy that leads to congestive heart failure. To test whether DOXO affects the cardiac progenitor cell (CPC) compartment, CPCs were exposed to increasing doses of the anthracycline for 12, 24 and 48 hours. At 0.1 μ M, DOXO did not alter CPC survival. However, 0.5 and 1 μ M DOXO reduced progressively and dramatically CPC viability. Apoptosis measured by TdT assay, DNA laddering and caspase-3 activity increased markedly with time peaking at 48 hours. The impact of DOXO on CPC growth was determined by BrdU and phospho-H3 labeling. The percentage of BrdU-positive CPCs and the mitotic index decreased with increasing doses of DOXO and time. The expression of the molecular regulators of G1 and G1\S transition, cyclin D1 and cdk4, decreased progressively with increasing concentration of DOXO and time, and these changes were coupled with a severe reduction in phospho-Rb in CPCs. Also, DOXO resulted in a transient increase of the cell cycle inhibitor p21 Cip and a persistent increase in p16 INK4a in CPCs. To determine whether the generation of reactive oxygen species (ROS) was responsible for DNA damage in CPCs, the presence of 8-OH-deoxyguanosine (8-OHdG) was measured. DOXO treatment strikingly increased the fraction of CPCs positive for 8-OHdG. Moreover, the expression of the antioxidant enzymes manganese superoxide dismutase and catalase failed to increase and counteract ROS-mediated DNA damage. Severe telomeric shortening occurred in CPCs exposed to DOXO. Telomere attrition was also favored by a significant decrease in telomerase activity in DOXO-treated CPCs. Dysfunctional telomeres trigger a DNA damage response in which the major determinant is the expression of p53. Both the transcription factor p53 and its target gene p21 Cip1 were upregulated in DOXO-treated CPCs. These molecular adaptations of CPCs increased with the dose and time of exposure to the anthracycline. In conclusion, these observations support the notion that DOXO-induced heart failure is a stem cell cardiomyopathy.